Synucleinopathies are a group of neurodegenerative diseases, which includes Parkinson's disease (PD), that are characterized by the formation of insoluble cytoplasmic inclusions of alpha-synuclein (alpha-syn).1 PD is an especially relevant therapeutic target is the second most common neurodegenerative disease and exceeds 5.6 billion dollars in economic costs each year.2-4 There is significant evidence that the toxic species in these diseases precedes aggregate or fibril formation, manifesting as a soluble oligomeric species.5 This proposal describes a unique multi-technique approach to analyzing alpha-syn oligomer formation in yeast and neuronal cells in order to identify the role of oligomers in synucleinopathy toxicity. To achieve this goal, I will investigate early oligomer formation in live cells using a multiple in vivo approaches (Bimolecular Fluorescence Complementation6, 7 and Fluorescence Lifetime Imaging Microscopy8) and I will correlate these data with changes in oligomer populations using methods that employ oligomer separation by electrophoresis (Aim 1). Using these complementary approaches to assessing oligomer populations (Aim 1), I will investigate how recently identified suppressors and enhancers of alpha-syn toxicity alter the alpha-syn oligomer populations in both yeast (Aim 2) and neuronal cells (Aim 3). I anticipate these studies will yield novel information about the role of specific alpha-syn oligomer assemblies in cellular toxicity. The information from these studies is significant for the development of targeted therapeutics for the cellular toxicity associated with synucleinopathies as well as understanding, in a broader context, the pathway of oligomer toxicity in amyloid diseases. The experiments outlined in this proposal will resolve critical information about the toxic alpha-synuclein oligomer in synucleinopathies, which includes Parkinson's disease (PD). PD is the second most common neurodegenerative disease, affecting over 1.5 million people in the United States alone.2-4 Currently, there is no cure or effective therapy for PD and the results from these studies will be essential in the development of therapeutics that target this toxic oligomer species and halt disease progression.